Top2 and Sgs1-Top3 Act Redundantly to Ensure rDNA Replication Termination.

Kamilla Mundbjerg,Lotte Bjergbaek,Signe W Jørgensen,Iben Bach Bentsen,Jakob Madsen Pedersen,Michael Lisby,Ida Nielsen,Jacob Fredsøe,Anni H Andersen,Gregory P Copenhaver

doi:10.1371/journal.pgen.1005697

Kamilla Mundbjerg, Lotte Bjergbaek + Show 8 more

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https://doi.org/10.1371/journal.pgen.1005697

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Abstract

Faithful DNA replication with correct termination is essential for genome stability and transmission of genetic information. Here we have investigated the potential roles of Topoisomerase II (Top2) and the RecQ helicase Sgs1 during late stages of replication. We find that cells lacking Top2 and Sgs1 (or Top3) display two different characteristics during late S/G2 phase, checkpoint activation and accumulation of asymmetric X-structures, which are both independent of homologous recombination. Our data demonstrate that checkpoint activation is caused by a DNA structure formed at the strongest rDNA replication fork barrier (RFB) during replication termination, and consistently, checkpoint activation is dependent on the RFB binding protein, Fob1. In contrast, asymmetric X-structures are formed independent of Fob1 at less strong rDNA replication fork barriers. However, both checkpoint activation and formation of asymmetric X-structures are sensitive to conditions, which facilitate fork merging and progression of replication forks through replication fork barriers. Our data are consistent with a redundant role of Top2 and Sgs1 together with Top3 (Sgs1-Top3) in replication fork merging at rDNA barriers. At RFB either Top2 or Sgs1-Top3 is essential to prevent formation of a checkpoint activating DNA structure during termination, but at less strong rDNA barriers absence of the enzymes merely delays replication fork merging, causing an accumulation of asymmetric termination structures, which are solved over time.

Highlights

During DNA replication termination two replication forks coming from opposite directions merge to form two fully replicated sister chromatids
Replication termination is the final step of the replication process, where the two replication forks converge and merge to form fully replicated sister chromatids
We demonstrate that the cell has two redundant pathways to overcome topological problems during rDNA replication termination, one involving Top2 and the other involving the RecQ helicase Sgs1, in concert with Top3

Summary

Introduction

During DNA replication termination two replication forks coming from opposite directions merge to form two fully replicated sister chromatids. The Tus-Ter sites are organized so that they form a trap for replication forks, thereby ensuring termination in a region opposite oriC in the circular E. coli genome [1]. In S. cerevisiae the rDNA locus holds the Replication Fork Barrier sequence (RFB). This barrier binds the Fob protein, which mediates polar fork stalling at RFB, resulting in replication termination in this region [2]. In S. pombe polar replication fork barriers are found both at the rDNA and mating type loci, where replication fork arrest occurs at the termination sites TER1-3 and RFP4 [3] and at the Replication Termination Sequence 1 (RTS1) [4], respectively. At yeast barriers members belonging to the Pif family helicases, Rrm and Pif in S. cerevisiae and Pfh in S. pombe, have been demonstrated to play profound roles for fork stalling and fork merging [5,6,7]

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